Wild-Type P53-Induced Phosphatase 1 (Wip1) Prevents Cellular Senescence At Physiological Oxygen Levels By Regulating Dna Damage Signaling During Dna Replication

Wip1 (PPM1D) is a nuclear serine/threonine protein phosphatase that is induced by p53 following the activation of DNA damage response (DDR) signaling. Upon repair of damage, Wip1 phosphatase activity dampens stress signaling and facilitates the return to homeostasis, thus, regulating recovery from DDR activation. Wip1 is amplified in several types of human primary cancer and has been shown to promote tumorigenesis by inhibiting several tumor suppressors, including p53, therefore its oncogenic potential is now recognized. Interestingly, while Wip1 knockout (Ppm1d–/–) mice are resistant to tumorigenesis, they exhibit reproductive and immune system defects, increased stress sensitivity and a moderately reduced lifespan. Furthermore, Ppm1d–/– Mouse Embryonic Fibroblasts (MEFs) exhibit premature senescence under conventional culture conditions. Little is known, however, about the mechanism by which the deletion of Ppm1d induces premature senescence. Therefore, we have investigated the role of Wip1 in the regulation of cellular senescence in MEFs. We found that at physiological oxygen tension (3% oxygen), early passage Ppm1d–/– MEFs underwent increased cellular senescence as compared with wild–type (Wt) MEFs, as shown by reduced proliferation rate, increased number of flattened enlarged cells, increased SA–β–Gal positive cells, and reduced BrdU incorporation. Moreover, under 3% oxygen, early passage Ppm1d–/– MEFs exhibited more γ–H2AX positive foci, increased activation of p53 and increased levels of p21 than Wt MEFs. Interestingly, we found that the increased levels of γ–H2AX in Ppm1d–/– MEFs were primarily associated with S–phase cells. Given that S–phase cells are more susceptible to oxidative damage, the increased levels of γ–H2AX may result from increased production of reactive oxygen species (ROS) in Ppm1d–/– MEFs. However, primary passage Wt and Ppm1d–/– MEFs cultured under 3% oxygen exhibited no significant difference in intracellular–ROS levels. Ppm1d–/– MEFs did show sustained activation of DDR signaling (γ–H2AX, p–p53Ser15, p21) after H2O2 treatment compared with Wt MEFs. Furthermore, Ppm1d–/– MEFs exhibited increased activation of Chk1, indicating that there is increased replicative stress signaling. These findings suggest that Wip1 prevents the induction of cellular senescence by regulating both oxidative stress– and replicative stress–induced DNA damage signaling during DNA replication. Citation Format: Hiroyasu Sakai, Christophe E. Redon, William M. Bonner, Ettore Appella, Sharlyn Mazur. Wild-type p53-induced phosphatase 1 (Wip1) prevents cellular senescence at physiological oxygen levels by regulating DNA damage signaling during DNA replication. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4041. doi:10.1158/1538-7445.AM2013-4041